Targeting VEGF-B as a novel treatment for insulin resistance and type 2 diabetes.

The prevalence of type 2 diabetes is rapidly increasing, with severe socioeconomic impacts. Excess lipid deposition in peripheral tissues impairs insulin sensitivity and glucose uptake, and has been proposed to contribute to the pathology of type 2 diabetes. However, few treatment options exist that directly target ectopic lipid accumulation. Recently it was found that vascular endothelial growth factor B (VEGF-B) controls endothelial uptake and transport of fatty acids in heart and skeletal muscle. Here we show that decreased VEGF-B signalling in rodent models of type 2 diabetes restores insulin sensitivity and improves glucose tolerance. Genetic deletion of Vegfb in diabetic db/db mice prevented ectopic lipid deposition, increased muscle glucose uptake and maintained normoglycaemia. Pharmacological inhibition of VEGF-B signalling by antibody administration to db/db mice enhanced glucose tolerance, preserved pancreatic islet architecture, improved ß-cell function and ameliorated dyslipidaemia, key elements of type 2 diabetes and the metabolic syndrome. The potential use of VEGF-B neutralization in type 2 diabetes was further elucidated in rats fed a high-fat diet, in which it normalized insulin sensitivity and increased glucose uptake in skeletal muscle and heart. Our results demonstrate that the vascular endothelium can function as an efficient barrier to excess muscle lipid uptake even under conditions of severe obesity and type 2 diabetes, and that this barrier can be maintained by inhibition of VEGF-B signalling. We propose VEGF-B antagonism as a novel pharmacological approach for type 2 diabetes, targeting the lipid-transport properties of the endothelium to improve muscle insulin sensitivity and glucose disposal.

Vascular endothelial growth factor B controls endothelial fatty acid uptake.

The vascular endothelial growth factors (VEGFs) are major angiogenic regulators and are involved in several aspects of endothelial cell physiology. However, the detailed role of VEGF-B in blood vessel function has remained unclear. Here we show that VEGF-B has an unexpected role in endothelial targeting of lipids to peripheral tissues. Dietary lipids present in circulation have to be transported through the vascular endothelium to be metabolized by tissue cells, a mechanism that is poorly understood. Bioinformatic analysis showed that Vegfb was tightly co-expressed with nuclear-encoded mitochondrial genes across a large variety of physiological conditions in mice, pointing to a role for VEGF-B in metabolism. VEGF-B specifically controlled endothelial uptake of fatty acids via transcriptional regulation of vascular fatty acid transport proteins. As a consequence, Vegfb(-/-) mice showed less uptake and accumulation of lipids in muscle, heart and brown adipose tissue, and instead shunted lipids to white adipose tissue. This regulation was mediated by VEGF receptor 1 and neuropilin 1 expressed by the endothelium. The co-expression of VEGF-B and mitochondrial proteins introduces a novel regulatory mechanism, whereby endothelial lipid uptake and mitochondrial lipid use are tightly coordinated. The involvement of VEGF-B in lipid uptake may open up the possibility for novel strategies to modulate pathological lipid accumulation in diabetes, obesity and cardiovascular diseases.

Imaging of a clinically relevant stroke model: glucose hypermetabolism revisited.

BACKGROUND AND PURPOSE: Ischemic stroke has been shown to cause hypermetabolism of glucose in the ischemic penumbra. Experimental and clinical data indicate that infarct-related systemic hyperglycemia is a potential therapeutic target in acute stroke. However, clinical studies aiming for glucose control in acute stroke have neither improved functional outcome nor reduced mortality. Thus, further studies on glucose metabolism in the ischemic brain are warranted. METHODS: We used a rat model of stroke that preserves collateral flow. The animals were analyzed by [2-(18)F]-2-fluoro-2-deoxy-d-glucose positron emission tomography or magnetic resonance imaging during 90-minute occlusion of the middle cerebral artery and during 60 minutes after reperfusion. Results were correlated to magnetic resonance imaging of cerebral blood flow, diffusion of water, lactate formation, and histological data on cell death and blood-brain barrier breakdown. RESULTS: We detected an increased [2-(18)F]-2-fluoro-2-deoxy-d-glucose uptake within ischemic regions succumbing to infarction and in the peri-infarct region. Magnetic resonance imaging revealed impairment of blood flow to ischemic levels in the infarct and a reduction of cerebral blood flow in the peri-infarct region. Magnetic resonance spectroscopy revealed lactate in the ischemic region and absence of lactate in the peri-infarct region. Immunohistochemical analyses revealed apoptosis and blood-brain barrier breakdown within the infarct. CONCLUSIONS: The increased uptake of [2-(18)F]-2-fluoro-2-deoxy-d-glucose in cerebral ischemia most likely reflects hypermetabolism of glucose meeting increased energy needs of ischemic and hypoperfused brain tissue, and it occurs under both anaerobic and aerobic conditions measured by local lactate production. Infarct-related systemic hyperglycemia could serve to facilitate glucose supply to the ischemic brain. Glycemic control by insulin treatment could negatively influence this mechanism.

Site-specifically 11C-labeled Sel-tagged annexin A5 and a size-matched control for dynamic in vivo PET imaging of protein distribution in tissues prior to and after induced cell death.

BACKGROUND: Radiolabeled annexin A5 (AnxA5) is widely used for detecting phosphatidylserine exposed on cell surfaces during apoptosis. We describe here a new method for labeling AnxA5 and a size-matched control protein with short-lived carbon-11, for probing the specificity of in vivo cell death monitoring using positron emission tomography (PET) imaging. METHODS: AnxA5 and the control protein were recombinantly expressed with a C-terminal "Sel-tag", the tetrapeptide -Gly-Cys-Sec-Gly-COOH. The proteins were then labeled either fluorescently for in vitro corroborations of binding behaviors or with 11C for dynamic in vivo PET studies. RESULTS: AnxA5 demonstrated retained calcium-dependent binding to apoptotic cells after the C-terminus modification. The control protein showed no functional binding. The 11C-ligands demonstrated similar in vivo pharmacokinetic behavior in healthy mice except for higher uptake in kidney and higher intact elimination to urine of AnxA5. After inducing hepatic apoptosis, however, the uptake of labeled AnxA5 in the targeted tissue increased compared to baseline levels while that of the control protein tended to decrease. CONCLUSIONS: These data suggest that the combined use of these two tracers can facilitate differentiating specific AnxA5 binding and its changes caused by induced cell death from uptake due to non-specific permeability and retention effects at baseline or after therapy. GENERAL SIGNIFICANCE: The Sel-tag enables rapid and mild reactions with electrophilic agents giving site-specifically labeled proteins for multi-probe analyses. The combined use of 11C-labeled AnxA5 and a size-matched control protein with dynamic PET can be useful for evaluating drug effects on target as well as off-target tissues.

Selective intra-arterial administration of ¹8F-FDG to the rat brain -- effects on hemispheric uptake.

INTRODUCTION: The purpose of this study was to investigate the radioligand uptake and iodine contrast distribution in the intra- and extracranial circulation of the rat, after intra-arterial injections to the common carotid artery and different parts of the internal carotid artery. METHODS: All animal experiments were carried out in accordance with Karolinska Institutet's guidelines and were approved by the local laboratory animal ethics committee. We used clinical neurointerventional systems to place microcatheters in the extra- or intracranial carotid artery of 15 Sprague-Dawley rats. Here, injection dynamics of iodine contrast was assessed using digital subtraction angiography. Maintaining the catheter position, the animals were placed in a micro PET and small-animal positron emission tomography (PET) was used to analyze injections [2-(18)F]-2-fluoro-2-deoxy-D-glucose ((18)F-FDG). RESULTS: Microcatheters had to be placed in the intracranial carotid artery (iICA) for the infusate to distribute to the brain. Selective injection via the iICA resulted in a 9-fold higher uptake of (18)F-FDG in the injected hemisphere (p < 0.005) compared to both intravenous and more proximal carotid artery injections. Furthermore, selective injection gave a dramatically improved contrast between the brain and extracranial tissue. CONCLUSION: Intra-arterial injection increases the cerebral uptake of a radiotracer dramatically compared to systemic injection. This technique has potential applications for endovascular treatment of malignancies allowing intra-interventional modifications of injection strategy, based on information on tumor perfusion and risk to surrounding normal parenchyma. Furthermore the technique may increase diagnostic sensitivity and avoid problems due to peripheral pharmacological barriers and first passage metabolism of labile tracers.

Radiolabelled proteins for positron emission tomography: Pros and cons of labelling methods.

BACKGROUND: Dynamic biomedical research is currently yielding a wealth of information about disease-associated molecular alterations on cell surfaces and in the extracellular space. The ability to visualize and quantify these alterations in vivo could provide important diagnostic information and be used to guide individually-optimized therapy. Biotechnology can provide proteinaceous molecular probes with highly specific target recognitions. Suitably labelled, these may be used as tracers for radionuclide-based imaging of molecular disease signatures. If the labels are positron-emitting radionuclides, the superior resolution, sensitivity and quantification capability of positron emission tomography (PET) can be exploited. SCOPE OF REVIEW: This article discusses different approaches to labelling proteins with positron-emitting nuclides with suggestions made depending on the biological features of the tracers. MAJOR CONCLUSIONS: Factors such as matching biological and physical half-lives, availability of the nuclide, labelling yields, and influences of labelling on targeting properties (affinity, charge and lipophilicity, cellular processing and retention of catabolites) should be considered when selecting a labelling strategy for each proteinaceous tracer. GENERAL SIGNIFICANCE: The labelling strategy used can make all the difference between success and failure in a tracer application. This review emphasises chemical, biological and pharmacological considerations in labelling proteins with positron-emitting radionuclides.

Radiolabelled receptor-tyrosine-kinase targeting drugs for patient stratification and monitoring of therapy response: prospects and pitfalls.

Transmembrane receptor tyrosine kinases (RTKs) are overexpressed in many malignancies. RTK signalling triggers cell proliferation, suppression of apoptosis, increased motility, and recruitment of neovasculature. Overexpressed RTKs are the molecular targets for an increasing number of anticancer drugs. Monoclonal antibodies block the ligands or their binding sites and prevent receptor dimerisation, thereby hindering RTK signalling. The antibody-dependent cellular cytotoxicity can boost the therapeutic effect. Small-molecule tyrosine kinase inhibitors (TKIs) hamper downstream RTK signalling by targeting the intracellular kinase domain. These drugs have significantly increased survival in several patient groups. Improved patient stratification and therapy monitoring might further enhance the efficacy of anti-RTK therapy. Radionuclide-based molecular imaging can provide methods for localising and estimating the expression of RTKs. It can potentially identify patients who have tumours that overexpress RTK and would, therefore, most likely benefit from a targeted treatment. Monitoring changes in RTK expression during therapy could help avoid overtreatment and undertreatment. Radionuclide-based methods are less invasive and less sensitive to expression heterogeneity than more conventional sampling methods. The biochemical information is also obtained in an anatomical context. The development of radiolabelled anti-RTK drugs and their analogues is the subject of intensive preclinical and translational research. In this review, we present current approaches to developing imaging probes for in-vivo RTK visualisation and discuss their advantages and disadvantages.

[68Ga]ABY-028: an albumin-binding domain (ABD) protein-based imaging tracer for positron emission tomography (PET) studies of altered vascular permeability and predictions of albumin-drug conjugate transport.

BACKGROUND: Albumin is commonly used as a carrier platform for drugs to extend their circulatory half-lives and influence their uptake into tissues that have altered permeability to the plasma protein. The albumin-binding domain (ABD) protein, which binds in vivo to serum albumin with high affinity, has proven to be a versatile scaffold for engineering biopharmaceuticals with a range of binding capabilities. In this study, the ABD protein equipped with a mal-DOTA chelator (denoted ABY-028) was radiolabeled with gallium-68 (68Ga). This novel radiotracer was then used together with positron emission tomography (PET) imaging to examine variations in the uptake of the ABD-albumin conjugate with variations in endothelial permeability. RESULTS: ABY-028, produced by peptide synthesis in excellent purity and stored at - 20 °C, was stable for 24 months (end of study). [68Ga]ABY-028 could be obtained with labeling yields of > 80% and approximately 95% radiochemical purity. [68Ga]ABY-028 distributed in vivo with the plasma pool, with highest radioactivity in the heart ventricles and major vessels of the body, a gradual transport over time from the circulatory system into tissues and elimination via the kidneys. Early [68Ga]ABY-028 uptake differed in xenografts with different vascular properties: mean standard uptake values (SUVmean) were initially 5 times larger in FaDu than in A431 xenografts, but the difference decreased to 3 after 1 h. Cutaneously administered, vasoactive nitroglycerin increased radioactivity in the A431 xenografts. Heterogeneity in the levels and rates of increases of radioactivity uptake was observed in sub-regions of individual MMTV-PyMT mammary tumors and in FaDu xenografts. Higher uptake early after tracer administration could be observed in lower metabolic regions. Fluctuations in the increased permeability for the tracer across the blood-brain-barrier (BBB) direct after experimentally induced stroke were monitored by PET and the increased uptake was confirmed by ex vivo phosphorimaging. CONCLUSIONS: [68Ga]ABY-028 is a promising new tracer for visualization of changes in albumin uptake due to disease- and pharmacologically altered vascular permeability and their potential effects on the passive uptake of targeting therapeutics based on the ABD protein technology.

On the selection of a tracer for PET imaging of HER2-expressing tumors: direct comparison of a 124I-labeled affibody molecule and trastuzumab in a murine xenograft model.

UNLABELLED: Human epidermal growth factor receptor type 2 (HER2) is a tyrosine kinase, which is often overexpressed in many carcinomas. Imaging HER2 expression in malignant tumors can provide important prognostic and predictive diagnostic information. The use of anti-HER2 tracers labeled with positron-emitting radionuclides may increase the sensitivity of HER2 imaging. The goal of this study was to compare directly 2 approaches for developing anti-HER2 PET tracers: a (124)I-labeled monoclonal antibody and a small (7-kDa) scaffold protein, the Affibody molecule. METHODS: The anti-HER2 Affibody Z(HER2:342) and humanized monoclonal antibody trastuzumab were labeled with (124/125)I using p-iodobenzoate (PIB) as a linker. Cellular processing of both tracers by HER2-expressing cells was investigated. The biodistributions of (124)I-PIB-Z(HER2:342) and (125)I-PIB-trastuzumab were compared in BALB/C nu/nu mice bearing HER2-expressing NCI-N87 xenografts using paired labels. Small-animal PET of (124)I-PIB-Z(HER2:342) and (124)I-PIB-trastuzumab in tumor-bearing mice was performed at 6, 24, and 72 h after injection. RESULTS: Both radioiodinated Z(HER2:342) and trastuzumab bound specifically to HER2-expressing cells in vitro and specifically targeted HER2-expressing xenografts in vivo. Radioiodinated trastuzumab was more rapidly internalized and degraded, which resulted in better retention of radioactivity delivered by Z(HER2:342). Total uptake of trastuzumab in tumors was higher than that of (124)I-PIB-Z(HER2:342). However, tumor-to-organ ratios were appreciably higher for (124)I-PIB-Z(HER2:342) due to the more rapid clearance of radioactivity from blood and normal organs. The ex vivo results were confirmed by small-animal PET. CONCLUSION: The use of the small scaffold targeting Affibody provides better contrast in HER2 imaging than does the monoclonal antibody.

Synthesis and preclinical evaluation of [(11)C]PAQ as a PET imaging tracer for VEGFR-2.

PURPOSE: (R,S)-N-(4-Bromo-2-fluorophenyl)-6-methoxy-7-((1-methyl-3-piperidinyl)methoxy)-4-quinazolinamine (PAQ) is a tyrosine kinase inhibitor with high affinity for the vascular endothelial growth factor receptor 2 (VEGFR-2), which plays an important role in tumour angiogenesis. The aim of this work was to develop and evaluate in mice the (11)C-labelled analogue as an in vivo tracer for VEGFR-2 expression in solid tumours. METHODS: [(11)C]PAQ was synthesized by an N-methylation of desmethyl-PAQ using [(11)C]methyl iodide. The tracer's pharmacokinetic properties and its distribution in both subcutaneous and intraperitoneal tumour models were evaluated with positron emission tomography (PET). [(18)F]FDG was used as a reference tracer for tumour growth. PET results were corroborated by ex vivo and in vitro phosphor imaging and immunohistochemical analyses. RESULTS: In vitro assays and PET in healthy animals revealed low tracer metabolism, limited excretion over 60 min and a saturable and irreversible binding. Radiotracer uptake in subcutaneous tumour masses was low, while focal areas of high uptake (up to 8% ID/g) were observed in regions connecting the tumour to the host. Uptake was similarly high but more distributed in tumours growing within the peritoneum. The pattern of radiotracer uptake was generally different from that of the metabolic tracer [(18)F]FDG and correlated well with variations in VEGFR-2 expression determined ex vivo by immunohistochemical analysis. CONCLUSION: These results suggest that [(11)C]PAQ has potential as a noninvasive PET tracer for in vivo imaging of VEGFR-2 expression in angiogenic "hot spots".

Irreversible inhibition of cytosolic thioredoxin reductase 1 as a mechanistic basis for anticancer therapy.

Cancer cells adapt to their inherently increased oxidative stress through activation of the glutathione (GSH) and thioredoxin (TXN) systems. Inhibition of both of these systems effectively kills cancer cells, but such broad inhibition of antioxidant activity also kills normal cells, which is highly unwanted in a clinical setting. We therefore evaluated targeting of the TXN pathway alone and, more specifically, selective inhibition of the cytosolic selenocysteine-containing enzyme TXN reductase 1 (TXNRD1). TXNRD1 inhibitors were discovered in a large screening effort and displayed increased specificity compared to pan-TXNRD inhibitors, such as auranofin, that also inhibit the mitochondrial enzyme TXNRD2 and additional targets. For our lead compounds, TXNRD1 inhibition correlated with cancer cell cytotoxicity, and inhibitor-triggered conversion of TXNRD1 from an antioxidant to a pro-oxidant enzyme correlated with corresponding increases in cellular production of H2O2 In mice, the most specific TXNRD1 inhibitor, here described as TXNRD1 inhibitor 1 (TRi-1), impaired growth and viability of human tumor xenografts and syngeneic mouse tumors while having little mitochondrial toxicity and being better tolerated than auranofin. These results display the therapeutic anticancer potential of irreversibly targeting cytosolic TXNRD1 using small molecules and present potent and selective TXNRD1 inhibitors. Given the pronounced up-regulation of TXNRD1 in several metastatic malignancies, it seems worthwhile to further explore the potential benefit of specific irreversible TXNRD1 inhibitors for anticancer therapy.

ß3-Adrenergically induced glucose uptake in brown adipose tissue is independent of UCP1 presence or activity: Mediation through the mTOR pathway.

OBJECTIVE: Today, the presence and activity of brown adipose tissue (BAT) in adult humans is generally equated with the induced accumulation of [2-18F]2-fluoro-2-deoxy-d-glucose ([18F]FDG) in adipose tissues, as investigated by positron emission tomography (PET) scanning. In reality, PET-FDG is currently the only method available for in vivo quantification of BAT activity in adult humans. The underlying assumption is that the glucose uptake reflects the thermogenic activity of the tissue. METHODS: To examine this basic assumption, we here followed [18F]FDG uptake by PET and by tissue [3H]-2-deoxy-d-glucose uptake in wildtype and UCP1(-/-) mice, i.e. in mice that do or do not possess the unique thermogenic and calorie-consuming ability of BAT. RESULTS: Unexpectedly, we found that ß3-adrenergically induced (by CL-316,243) glucose uptake was UCP1-independent. Thus, whereas PET-FDG scans adequately reflect glucose uptake, this acute glucose uptake is not secondary to thermogenesis but is governed by an independent cellular signalling, here demonstrated to be mediated via the previously described KU-0063794-sensitive mTOR pathway. CONCLUSIONS: Thus, PET-FDG scans do not exclusively reveal active BAT deposits but rather any tissue possessing an adrenergically-mediated glucose uptake pathway. In contrast, we found that the marked glucose uptake-ameliorating effect of prolonged ß3-adrenergic treatment was UCP1 dependent. Thus, therapeutically, UCP1 activity is required for any anti-diabetic effect of BAT activation.

The tyrosine kinase inhibitor PD153035: implication of labeling position on radiometabolites formed in vitro.

INTRODUCTION: The epidermal growth factor receptor is highly expressed in several types of cancers. Molecules with high affinity to its intracellular tyrosine kinase domain are being developed as in vivo imaging probes. The 4-anilinoquinazoline PD153035 has promising in vitro and in vivo properties for development as a reversible radioligand. Labeling it with carbon-11 in either of its two methoxy positions can potentially give rise to different radiometabolites and, consequently, different imaging capabilities. An evaluation of the radiotracers' metabolism was needed to determine the potential significance of the labeling position. METHODS: PD153035 was labeled in the 6- and 7-O-methoxy positions by reacting the corresponding O-desmethyl precursors with [(11)C]methyl iodide. The two radiolabeled compounds were each incubated for 1 h with human and rat liver microsomes. At five time points, the radiolabeled metabolites were examined using radio-liquid chromatography. One metabolite was isolated and subjected to mass spectroscopic analysis. RESULTS: A major polar metabolite was obtained in all incubations. Its molecular weight was consistent with an addition of oxygen, and its fragmentation was consistent with an N-oxidation rather than an aromatic hydroxylation. Regioselective 7-O-dealkylation was also observed, albeit in substantial amounts only in the assay using human microsomes. CONCLUSIONS: Radiolabeling in the 7-O-methoxy position is advocated, since the labeled metabolites produced in the 7-O-demethylation are polar and probably rapidly cleared. The differences observed in the incubations with rat and human microsomes suggest that in vivo positron emission tomography studies with (11)C-labeled PD153035 in rodents may not be directly predictive for studies in humans.

Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake.

BACKGROUND: Though overexpression of epidermal growth factor receptor (EGFR) in several forms of cancer is considered to be an important prognostic biomarker related to poor prognosis, clear correlations between biomarker assays and patient management have been difficult to establish. Here, we utilize a targeting directly followed by a non-targeting tracer-based positron emission tomography (PET) method to examine some of the aspects of determining specific EGFR binding in tumors. METHODS: The EGFR-binding Affibody molecule ZEGFR:2377 and its size-matched non-binding control ZTaq:3638 were recombinantly fused with a C-terminal selenocysteine-containing Sel-tag (ZEGFR:2377-ST and ZTaq:3638-ST). The proteins were site-specifically labeled with DyLight488 for flow cytometry and ex vivo tissue analyses or with (11)C for in vivo PET studies. Kinetic scans with the (11)C-labeled proteins were performed in healthy mice and in mice bearing xenografts from human FaDu (squamous cell carcinoma) and A431 (epidermoid carcinoma) cell lines. Changes in tracer uptake in A431 xenografts over time were also monitored, followed by ex vivo proximity ligation assays (PLA) of EGFR expressions. RESULTS: Flow cytometry and ex vivo tissue analyses confirmed EGFR targeting by ZEGFR:2377-ST-DyLight488. [Methyl-(11)C]-labeled ZEGFR:2377-ST-CH3 and ZTaq:3638-ST-CH3 showed similar distributions in vivo, except for notably higher concentrations of the former in particularly the liver and the blood. [Methyl-(11)C]-ZEGFR:2377-ST-CH3 successfully visualized FaDu and A431 xenografts with moderate and high EGFR expression levels, respectively. However, in FaDu tumors, the non-specific uptake was large and sometimes equally large, illustrating the importance of proper controls. In the A431 group observed longitudinally, non-specific uptake remained at same level over the observation period. Specific uptake increased with tumor size, but changes varied widely over time in individual tumors. Total (membranous and cytoplasmic) EGFR in excised sections increased with tumor growth. There was no positive correlation between total EGFR and specific tracer uptake, which, since ZEGFR:2377 binds extracellularly and is slowly internalized, indicates a discordance between available membranous and total EGFR expression levels. CONCLUSIONS: Same-day in vivo dual tracer imaging enabled by the Sel-tag technology and (11)C-labeling provides a method to non-invasively monitor membrane-localized EGFR as well as factors affecting non-specific uptake of the PET ligand.

A method for comparing intra-tumoural radioactivity uptake heterogeneity in preclinical positron emission tomography studies.

BACKGROUND: Non-uniformity influences the interpretation of nuclear medicine based images and consequently their use in treatment planning and monitoring. However, no standardised method for evaluating and ranking heterogeneity exists. Here, we have developed a general algorithm that provides a ranking and a visualisation of the heterogeneity in small animal positron emission tomography (PET) images. METHODS: The code of the algorithm was written using the Matrix Laboratory software (MATLAB). Parameters known to influence the heterogeneity (distances between deviating peaks, gradients and size compensations) were incorporated into the algorithm. All data matrices were mathematically constructed in the same format with the aim of maintaining overview and control. Histograms visualising the spread and frequency of contributions to the heterogeneity were also generated. The construction of the algorithm was tested using mathematically generated matrices and by varying post-processing parameters. It was subsequently applied in comparisons of radiotracer uptake in preclinical images in human head and neck carcinoma and endothelial and ovarian carcinoma xenografts. RESULTS: Using the developed algorithm, entire tissue volumes could be assessed and gradients could be handled in an indirect manner. Similar-sized volumes could be compared without modifying the algorithm. Analyses of the distribution of different tracers gave results that were generally in accordance with single plane preclinical images, indicating that it could appropriately handle comparisons of targeting vs. non-targeting tracers and also for different target levels. Altering the reconstruction algorithm, pixel size, tumour ROI volumes and lower cut-off limits affected the calculated heterogeneity factors in expected directions but did not reverse conclusions about which tumour was more or less heterogeneous. CONCLUSIONS: The algorithm constructed is an objective and potentially user-friendly tool for one-to-one comparisons of heterogeneity in whole similar-sized tumour volumes in PET imaging.

[11C]MADAM Used as a Model for Understanding the Radiometabolism of Diphenyl Sulfide Radioligands for Positron Emission Tomography (PET).

In quantitative PET measurements, the analysis of radiometabolites in plasma is essential for determining the exact arterial input function. Diphenyl sulfide compounds are promising PET and SPECT radioligands for in vivo quantification of the serotonin transporter (SERT) and it is therefore important to investigate their radiometabolism. We have chosen to explore the radiometabolic profile of [11C]MADAM, one of these radioligands widely used for in vivo PET-SERT studies. The metabolism of [11C]MADAM/MADAM was investigated using rat and human liver microsomes (RLM and HLM) in combination with radio-HPLC or UHPLC/Q-ToF-MS for their identification. The effect of carrier on the radiometabolic rate of the radioligand [11C]MADAM in vitro and in vivo was examined by radio-HPLC. RLM and HLM incubations were carried out at two different carrier concentrations of 1 and 10 µM. Urine samples after perfusion of [11C]MADAM/MADAM in rats were also analysed by radio-HPLC. Analysis by UHPLC/Q-ToF-MS identified the metabolites produced in vitro to be results of N-demethylation, S-oxidation and benzylic hydroxylation. The presence of carrier greatly affected the radiometabolism rate of [11C]MADAM in both RLM/HLM experiments and in vivo rat studies. The good concordance between the results predicted by RLM and HLM experiments and the in vivo data obtained in rat studies indicate that the kinetics of the radiometabolism of the radioligand [11C]MADAM is dose-dependent. This issue needs to be addressed when the diarylsulfide class of compounds are used in PET quantifications of SERT.

Changes in Regional Cerebral Oxidative Metabolism Induced by Tactile Learning and Recognition in Man.

We measured the regional cerebral oxidative metabolism (rCMRO2) with positron emission tomography in normal healthy volunteers in three different stages: rest, tactile learning, and tactile recognition of complicated geometrical objects. The frequency of manipulatory movements during tactile recognition was twice that of tactile learning. Tactile recognition with the right hand increased rCMRO2 in six prefrontal cortical areas, bilaterally in the supplementary motor areas, the premotor areas and supplementary sensory areas, in the left primary motor and primary sensory area, in the left anterior superior parietal lobule, bilaterally in the secondary somatosensory area, the anterior insula, lingual gyri, hippocampus, basal ganglia, anterior parasagittal cerebellum, and lobus posterior cerebelli. These structures have in other studies been found to participate in manipulatory movements and analysis of somatosensory information. Tactile learning increased rCMRO2 in the same structures as did tactile recognition. Thus we found no differences in the anatomical structures participating in storage and retrieval. However the rCMRO2 increases in the left premotor cortex, supplementary motor area, and left somatosensory hand area were larger during tactile recognition in accordance with the higher frequency of manipulatory movements and higher flux of somatosensory information from the periphery during recognition. Despite this the rCMRO2 was significantly higher in the neocerebellar cortex during tactile learning. Since there were no learning effects on the manipulatory movements, this extra metabolic activity in the lateral cerebellum was attributed to energy demanding processes associated with climbing fibre activity during storage of somatosensory information.

Somatosensory Discrimination of Shape: Tactile Exploration and Cerebral Activation.

This study of somatosensory discrimination of rectangular parallelepipeda with the right hand had three purposes: (i) to describe the exploratory finger movements; (ii) to reveal the anatomical brain structures specifically engaged in the production of exploratory finger movements; and (iii) to reveal the anatomical structures specifically engaged in the discrimination of tactually sensed shape. The thumb was the most active finger, moving with a mean exploration frequency of 2.4 Hz, as evident from videotape records of the exploratory finger movements. The cerebral structures activated during somatosensory discrimination were mapped by measurements of regional cerebral blood flow (rCBF) in six healthy male volunteers with positron emission tomography (PET) and the use of the computerized brain atlas of Greitz et al. (1991, J. Comp. Ass. Tomogr., 15, 26 - 38). The rCBF changes caused by somatosensory discrimination were compared point-to-point to a PET-study on right-hand finger movements and a PET-study on vibration stimulation of the right hand. From these results the following conclusions were drawn. The rCBF increase in the left superior parietal lobule indicated the site engaged in the analysis of shape. The rCBF increases in the left supplementary sensory area, bilaterally in premotor areas, in the left putamen, the right dentate nucleus and bilaterally in the posterior cerebellum were related to the control of the tactile exploratory finger movements. The rCBF increases in the right homologue of Broca's area, bilaterally in the superior prefrontal cortex and in the right midfrontal cortex probably resulted from working memory, the direction of attention, and the discrimination process.

Central representation of chronic ongoing neuropathic pain studied by positron emission tomography.

This study was undertaken to explore whether the neural substrates demonstrated in brain imaging studies on experimentally induced pain are involved in the perception of chronic neuropathic pain. We investigated the cerebral representation of chronic lateralised ongoing pain in patients with painful mononeuropathy (PMN, i.e., pain in the distribution of a nerve, neuralgia) with positron emission tomography (PET), using regional cerebral blood flow (rCBF) as an index for neuronal activity. Eight patients (29-53 years) with PMN in the lower extremity (4 in the right, 4 in the left) were recruited. Paired comparisons of rCBF were made between the patient's habitual pain (HP) state and the pain alleviated (PA) state following a successful regional nerve block (RNB) with lidocaine. The ongoing neuropathic pain resulted in activation of bilateral anterior insula, posterior parietal, lateral inferior prefrontal, and posterior cingulate cortices as well as the posterior sector of the right anterior cingulate cortex (ACC), Brodmann area (BA) 24, regardless of the side of PMN. In addition, a reduction in rCBF was noted in the contralateral posterior thalamus. No significant change of rCBF was detected in the somatosensory areas, i.e., SI and SII. The cerebral activation pattern, while addressing the differences between the HP and PA states, emphasises the affective-motivational dimension in chronic ongoing neuropathic pain. The striking preferential activation of the right ACC (BA 24), regardless of the side of the PMN, not only confirms that the ACC participates in the sensorial/affectional aspect of the pain experience but also suggests a possible right hemispheric lateralisation of the ACC for affective processing in chronic ongoing neuropathic pain. Our data suggests that the brain employs different central mechanisms for chronic neuropathic pain and experimentally induced acute pain, respectively.

Traumatic nociceptive pain activates the hypothalamus and the periaqueductal gray: a positron emission tomography study.

The study was conducted to investigate which areas of the brain respond to a painful encounter of minor dermal injury (a model of clinical pain) elicited by intracutaneous injection of a minute amount of ethanol. Four healthy volunteers (27-46 years) were subjected to positron emission tomographic (PET) investigation of regional cerebral blood flow (rCBF), using [15O]butanol as tracer. The ethanol (20 microliters, 70%) and saline (20 microliters, 0.9%) were injected intracutaneously 3 times in a single-blinded, semi-randomised manner for the pain experiment. All the injections were performed, adjacent to each other, at the lateral aspect of the right upper arm. Subjective sensory intensity of pain, unpleasantness and anxiety were rated with separate 100-mm visual analogue scales together with the Spielberger's State Anxiety Inventory (Spielberger et al. 1970) and heart rate. Paired-subtraction (pixel-by-pixel) between ethanol and saline was performed. Traumatic pain significantly caused higher ratings of intensity and affect scales, i.e., pain intensity, unpleasantness and increased sympathetic activity (evidenced by tachycardia). In contrast the anxiety rating remained unchanged. Acute traumatic nociceptive pain prominently activated the hypothalamus and periaqueductal gray (PAG). In addition, activations of the prefrontal cortex (PFC), insular, anterior cingulate cortex (ACC), posterior parietal cortex (PPC), primary motor/somatosensory areas (MI/SI: face, upper arm), supplementary motor area (SMA), and cerebellum were also demonstrated. The central processing of the pain-relevant/anticipatory arousal also engaged the PAG. This study demonstrates the involvement of the human cerebral cortex in perception, arousal, cognitive evaluative processes, and, hence, affective reactions (somatic/ autonomic outflow) associated with pain. The pain stimulus of traumatic character may, by its very nature, evoke the central processing to involve both the hypothalamus and the PAG.